Bile- and bile acid-induced genes of Clonorchis sinensis metacercaria analyzed using oligonucleotide

초록

Clonorchis sinensis metacercariae (CsMC) excyst in duodenum and migrate into bile duct. Bile is a strong stimulant promoting maturation and chemotactic behavior of the C. sinensis newly excysted juvenile (CsNEJ). To analyze gene expression profile of CsNEJ induced by bile and bile acids, 12K oligonucleotide microarray was fabricated loading 11,486 probes designed from an EST pool of C. sinensis adult and metacercaria. The CsMC were excysted with 0.01% trypsin and incubated in bile, cholic acid (CA) or lithocholic acid (LCA). Total RNA was extracted and genomic DNA was removed by DNase I treatment. The RNA samples were labeled with Cy5 fluorescent and hybridized to the microarrays. Each group was biologically triplicated. Scanned signals were normalized by global normalization method. Gene expression profiles of CsNEJ were dependent on incubation-time in bile. Up-regulated genes at early incubation period were ariadne homolog 2, protein phosphatase methylesterase-1, serine protease inhibitor B3, etc. These proteins were associated with initiation of CsNEJ growth. After middle bile-incubation period, energy metabolism related genes (UDP-glucose dehydrogenase, hydroxypyruvate isomerase, galactosidase beta 1, phosphate cytidylyltransferase, etc.) and growth related genes (phosphate cytidylyltransferase 2 ethanolamine, ariadne homolog 2, etc.) were up-regulated. These gene expressions were related development and migration of CsNEJ. Serine protease inhibitor B3, cysteine protease 6, myoglobin, gynecophoral canal protein were up-regulated by CA. It is associated with development of CsNEJ at early infection stage. Metabolism, signaling, and regulation related genes were downregulated by LCA. It was suggested LCA may inhibit cell activity of the CsNEJ.